It is now clear that epigenetic alterations including hypermethylation of gene promoters and some regions internal to a gene are consistent and early events in neoplastic progression. Such alterations are thought to contribute to the neoplastic process by transcriptional silencing of tumor suppressor gene expression, and by increasing the rate of genetic mutation. DNA methylation is reversible, since it does not alter the DNA sequence; however, it is heritable from cell to cell. As such, methylated genes could serve as biomarkers such as for early detection of cancer, risk assessment, predicting and monitoring response to therapy, and early detection of relapse.
Tumor DNA can be found in various body fluids and these fluids can potentially serve as diagnostic material. Evaluation of tumor DNA in these fluids requires methods that are specific as well as sensitive. A PCR-based technique called methylation-specific PCR (MSP) is known to detect one copy of methylated genomic DNA in one-thousand unmethylated copies of genomic DNA. Quantitative real time PCR (Q-PCR) allows a highly sensitive quantification of transcriptional levels or levels of the DNA of the gene of interest in a few hours with minimal handling the samples. cDNA or genomic copies of the gene of interest are quantitated by detecting PCR products as they accumulate using an optically detectable polynucleotide probe. Quantitative MSP (Q-MSP) allows highly sensitive detection of gene promoter methylation levels by real time PCR with methylation-specific primers probes.
Quantitative and multiplex MSP techniques have been modified in order to co-amplify several genes simultaneously in a nested or multiplex MSP assay to develop quantitative multiplex methylation-specific PCR (QM-MSP). The QM-MSP method is based on real-time PCR that uses one or more distinguishable optically detectable probes to increase the assay specificity and the sensitivity such that one to ten copies of the desired methylated gene among 100,000 unmethylated copies of the gene can be detected.
Studies from the inventors have shown that a panel of methylation markers can detect 100% of all breast carcinomas and 95% of DCIS, and cancer in cells from ductal fluid and in spontaneous nipple discharge. However, reproducible detection of methylated DNA from serum/plasma proved to be more difficult. Many studies in the past 15 years have reported the use of a single or a panel of markers to detect breast cancer in serum/plasma. However, no clinical validation studies have followed these preliminary findings, suggesting that there were technical difficulties, yet to be resolved, in this analysis: 1) First, the amount of methylated tumor DNA shed in the serum is a very small fraction of the total unmethylated DNA (either matching gene-specific DNA or a reference DNA such as actin) shed by normal cells. There is a disproportion between the relative abundance of the unmethylated or reference DNA and the rarity of the target methylated DNA in body fluids. This leads to lack of robustness of the assay due to masking of methylated signals by the more abundant species; 2) The extent of shedding of normal unmethylated DNA fluctuates daily and with various clinical states, such as surgery or infection; if the reference fluctuates, then inaccuracies occur in interpretation of % methylation of a target gene of interest; 3) The most sensitive assays available utilize nested PCR (with or without multiplex) to pre-amplify amplicons of target and reference DNA using external primers outside the region of interest prior to round two quantitative PCR. But there are technical limitations such that the target and reference DNA regions need to be co-amplified in the pre-amplification PCR reaction with the same efficiency, ideally using the same pair of external primers in order to maintain the accurate starting ratio of target to reference DNA. In nested PCR Actin or other genes cannot accurately serve as a reference for the gene of interest because of differences in efficiencies of primer hybridization between the non-identical genes. Thus the ratio of target to reference changes with each cycle of pre-amplification, depending on the relative differences in efficiency of hybridization of the DNA-specific primers to the target and reference DNAs; 4) Existing serum MSP assays usually require 10-20 ng genomic DNA per assay to test one gene one time. Since the level of total DNA in serum of cancer patients is low [median 65.4 ng/ml; range 6.3-268.6 ng/ml; Holdenrieder et al., Ann. N.Y. Acad. Sci. 1137:162-170 (2008)] assays of several genes require a significant amount of serum, making repeat studies problematic and clinical validation difficult because archival sera from existing clinical studies are available only in small volumes.